-
CTMI (2006) 309:189219c Springer-Verlag Berlin Heidelberg
2006
Rotavirus Proteins: Structure and Assembly
J. B. Pesavento1 S. E. Crawford2 M. K. Estes2 B. V. Venkataram
Prasad1 ()1Verna and Marrs McLean Department of Biochemistry and
Molecular Biology,Baylor College of Medicine, Houston, TX 77030,
[email protected] of Molecular Virology and
Microbiology, Baylor College of Medicine,Houston, TX 77030, USA
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . 190
2 Rotavirus Proteins . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . 192
3 Capsid Architecture . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . 1923.1 VP7 Layer and VP4 Spikes .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1933.2 Aqueous Channels . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . 1953.3 VP6 Layer . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . 1963.4 VP2 Layer and Transcription Enzyme Complex . . . . . .
. . . . . . . . . . . . . 1963.5 Genome Organization . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
4 Reassortants . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . 199
5 Protease-Enhanced Infectivity . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . 1995.1 Trypsin-Induced Unique
Order-to-Disorder Transition in the Spike . . . . . 200
6 Cell Entry . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . 2026.1 Possible Structural
Alterations in VP4 During Cell Entry . . . . . . . . . . . .
2036.1.1 Is the VP4 Spike a Trimer? . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . 2036.1.2 pH-Induced Changes of
the Spike:
Implication for Cell Entry and Antibody Neutralization . . . . .
. . . . . . . . 203
7 Endogenous Transcription . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . 204
8 Genome Replication and Packaging . . . . . . . . . . . . . . .
. . . . . . . . . . . . 2058.1 NSP3 and Genome Translation . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . 2068.2 NSP2
and NSP5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . 2068.3 A Working Model for Genome
Encapsidation in Rotavirus . . . . . . . . . . . 208
9 Maturation and Release . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . 209
10 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . 210
References . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . 211
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190 J. B. Pesavento et al.
Abstract Rotavirus is a major pathogen of infantile
gastroenteritis. It is a large andcomplexviruswith amultilayered
capsidorganization that integrates thedeterminantsof host
specicity, cell entry, and the enzymatic functions necessary for
endogenoustranscription of the genome that consists of 11 dsRNA
segments. These segmentsencode six structural and six nonstructural
proteins. In the last few years, there hasbeen substantial progress
in our understanding of both the structural and functionalaspects
of a variety of molecular processes involved in the replication of
this virus.Studies leading to this progress using of a variety of
structural and biochemicaltechniques including the recent
application of RNA interference technology haveuncovered several
unique and intriguing features related to viral morphogenesis.
Thisreview focuses on our current understanding of the structural
basis of the molecularprocesses that govern the replication of
rotavirus.
1Introduction
Rotavirus is a major cause of gastroenteritis in young children
(under age 5)worldwide. It is responsible for an estimated
600,000870,000 annual deathsworldwide
(Cohen2001;Kapikian2002;MidthunandKapikian1996;Parasharet al.
2003). Deaths from rotavirus are most prevalent in developing
nations,where patients may not always receive adequate medical
attention quicklyenough. Rotavirus infection occurs primarily in
the differentiated enterocytesof the jejunum in the small
intestine, which are responsible for digestion andabsorption (Moon
1994). Destruction of these cells results in the loss ofnutrient
and water absorption, followed by dehydration and malnutritionthat
ultimately can lead to death. An increasing number of reports
indicatethat rotavirus escapes the gastrointestinal tract resulting
in antigenemia inchildren and viremia in animal models (Blutt et
al. 2003) and the detection ofrotavirus antigen or RNA in tissues
of infected children and adults (Cioc andNuovo 2002; Hongou et al.
1998; Iturriza-Gomara et al. 2002; Lynch et al. 2001,2003; Morrison
et al. 2001; Pager et al. 2000). The full clinical signicance
ofsuch extraintestinal virus remains to be determined.
Rotavirus is amember of theReoviridae family, which consists of
11 genera(Fields 1996). Members of this family of viruses have
multilayered, nonen-veloped, icosahedral capsids with a diameter
ranging from approximately 600to 1000 . Each member of this family
encapsidates between 1012 segmentsof dsRNA. In these viruses, the
enzymatic machinery necessary for transcrip-tion is housed within
an intact core, where the genome is transcribed. Tran-scriptionally
active particles of these viruses are capable of repeated cycles
oftranscription. These viruses replicate in the cytoplasm of the
cell and encodeseveral nonstructural proteins to aid in their
replication and morphogenesisinside the host cell.
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Rotavirus Proteins: Structure and Assembly 191
Biochemical studies on rotaviruses have established much of our
basic un-derstanding of rotavirus infectivity, genome
transcription, morphogenesis,and viruscell interactions. The lack
of a reverse genetics system for rotavirus,as for all members of
the Reoviridae, has hampered a detailed understandingof the
intracellular functional roles of the virally encoded proteins. In
lieuof this, recombinant proteins and virus-like particles (VLPs)
have been veryuseful, not only in rotavirus but also in other dsRNA
viruses, for the under-standing of both biochemical and structural
properties of rotaviral structuraland nonstructural proteins. All
rotaviral genes of several rotavirus strainshave been cloned (Estes
and Cohen 1989). These genes have been successfullyexpressed, and
co-expressionof specic structural proteins has been shown toresult
in the spontaneous formation of virus-like particles (VLPs) and
otherfunctional complexes (Cohen et al. 1989; Crawford et al. 1994;
Estes et al.1987; Labbe et al. 1991; Mattion et al. 1991, 1992;
Zeng et al. 1994). In parallel,structural studies have played an
important role to help understand the virusfunctions in the context
of the three-dimensional structures of the virus andvirus-encoded
individual proteins. An exciting development in the eld ofrotavirus
biology in recent years is the application of RNA interference
tech-niques to study the functional roles of rotaviral proteins
during the processof infection (Arias et al. 2004; Campagna et al.
2005; Dector et al. 2002; Lopezet al. 2005; Silvestri et al.
2004).
Until recently, much of our understanding of the
structurefunction rela-tionships in rotaviruses has come from using
electron cryomicroscopy (cryo-EM) techniques (Prasad and Estes
2000). Determination of the overall low-resolution structure of
rotavirus using cryo-EM techniques in 1988 (Prasadet al. 1988)
began paving the way for more elaborate structural
characteri-zation of this virus (Prasad et al. 1990, 1996; Shaw et
al. 1993; Yeager et al.1990, 1994). In addition to providing a
detailed description of the architec-tural features of this large
and complex virus, including the topographicallocations of all the
structural proteins and their stoichiometric proportions,these
structural studies using cryo-EM techniques also providedmore
insightinto some of the biological functions of the virus such as
trypsin-enhancedinfectivity (Crawford et al. 2001), cell entry
(Dormitzer et al. 2004, Pesaventoet al. 2005), antibody
interactions (Prasad et al. 1990; Tihova et al. 2001), en-dogenous
transcription (Lawton et al. 1997a, 2000), and genome
organization(Pesavento et al. 2001, 2003b).
More recently, X-ray crystallography has been successfully
applied to de-termine the atomic structures of several of the
structural and nonstructuralproteins of rotavirus (Deo et al. 2002;
Dormitzer et al. 2002, 2004; Groft andBurley 2002; Jayaram et al.
2002; Mathieu et al. 2001). With the lack of anX-ray structure of
the rotavirus particle or any of its subassemblies, cryo-EM
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192 J. B. Pesavento et al.
reconstructions in combination with X-ray structural information
have lledthe void to some extent and provided more in-depth
structural characteri-zation of the particles at atomic resolution
(Dormitzer et al. 2004; Mathieuet al. 2001). With the spectacular
success in determining near atomic resolu-tion structures of the
bluetongue virus (BTV) core (Grimes et al. 1998) andorthoreovirus
core (Reinisch et al. 2000), there is the expectation that
theentire rotavirus or homologous subassemblies of rotavirus can be
addressedusing X-ray crystallography. The status of our current
understanding of thethree-dimensional structure of this important
medical pathogen and someof its proteins in the context of its
replication cycle is the main focus of thisreview.
2Rotavirus Proteins
The 11 dsRNA segments of the rotavirus genome code for six
structural andsix nonstructural proteins (Fig. 1a). The naming of
the structural proteinsis based on their molecular weights, with
VP1, the largest at 125 kDa, andVP8*, one of the two proteolytic
fragments of VP4, the smallest at 28 kDa. Thesix structural
proteins form the multi-layered capsid of the mature
rotavirusparticle. The nonstructural proteins, except for NSP1, are
essential for virusreplication. NSP1 is an RNA-binding protein that
directly interacts with IRF-3(Graff et al. 2002). The loss
ofNSP1doesnot seemtonegatively affect rotavirusreplication in
cultured cells (Silvestri et al. 2004). However, it plays a role
inpathogenesis in some animal models (reviewed in Desselberger
1997), likelyby antagonizing the type I interferon response to
increase viral pathogenesis(Barro and Patton 2005). In this regard,
NSP1 shares some similarities withNS1 of inuenza virus, although
the mechanism of action appears to beunique. The function and roles
that the rest of the rotaviral proteins play in thestructure and
replication of rotavirus are discussed below.Abrief summary ofthe
properties of the rotavirus structural and nonstructural proteins
is givenin Table 1.
3Capsid Architecture
The architectural features of the mature rotavirus along with
the positionsof various structural proteins are shown in Fig. 1b
and c. The mature infec-tious rotavirus particle 1000 in diameter
(including the spikes), is made ofthree concentric icosahedral
protein layers that encapsidate the genome of
-
Rotavirus Proteins: Structure and Assembly 193
Fig. 1ac a PAGE showing rotavirus RNA segments and geneprotein
assignments.The RNA segments are numbered in order of gel migration
on the left and theirencoded protein products are indicated on the
right. Gene segments 7, 8, and 9 arevery close in length and tend
to migrate nearly on top of one another. Gene 11 isalternatively
processed to produce NSP5 and NSP6. (Torres-Vega et al. 2000;
Welchet al. 1989). For protein molecular weights, see Table 1. b
Surface representation of themature rotavirus particle (TLP).
Arrows indicate the three types of aqueous channels,labeled I, II,
and III. The 60 VP4 spikes are colored red and the 780 copies of
VP7forming the outer capsid layer are shown in yellow. (Adapted
from Pesavento et al.2003b). c Cut-away of the TLP structure
showing the internal structural features. Thedensity due to genomic
RNA is removed for clarity. The internal VP6 protein layer isin
blue and the core VP2 layer in green. The ower-shaped VP1VP3
transcriptioncomplex is attached to the inside of the VP2 layer at
the ve-fold icosahedral axesdirectly below the type I channels and
is colored red. (Adapted fromPrasad et al. 1996)
11 dsRNA segments. The complete virion is called a
triple-layered particle(TLP). Like many of the members of the
Reoviridae, the capsid architectureis predominantly based on T=13
icosahedral symmetry.
3.1VP7 Layer and VP4 Spikes
The outer layer of the TLP is composed of two structural
proteins: VP7 andVP4. VP7, the major constituent of the outer
layer, is a glycoprotein in mostrotavirus strains although
glycosylation is not required for capsid assembly(Estes 2001).
Seven hundred eighty copies of VP7 are grouped as 260 trimersat all
the icosahedral and local three-fold axes of a T=13 icosahedral
latticesurrounding 132 channels. The outer layer is decorated by 60
spikes, each ofwhich is formed by a dimer of VP4 (Fig. 1b). Thus
each rotavirus particle has120 copies of VP4. The composition of
the spike was conrmed by cryo-EM
-
194 J. B. Pesavento et al.
Tabl
e1
Prop
erties
ofro
tavi
russtru
ctur
alan
dno
nstruc
tura
lpro
tein
sa
Gen
ePr
otein
Mas
sPo
st-tra
nslation
alLo
cation
Func
tion
alse
gmen
t(k
Da)
bm
odi
cation
(s)
(no.
ofco
pies
)pr
oper
ties
1VP1
125
SL
P(1
2)RNA-d
epen
dent
RNA
poly
mer
ase,
RNA
bind
ing,
intera
ctswith
VP2
and
VP3
2VP2
95Cleav
edSL
P(1
20)
RNA
bind
ing,
intera
ctswith
VP1
3VP3
88
SLP
(12)
Gua
nylyla
ndm
ethy
ltra
nsfera
se,
ssRNA
bind
ing,
intera
ctswith
VP1
4VP4
(VP5
*+
VP8
*)
85 (58+
27)
Cleav
edTLP
(120
)Hem
agglut
inin
,neu
tralizat
ion
antige
n,vi
rulenc
e,pr
otea
se-e
nhan
ced
infect
ivity,
cell
atta
chm
ent,
fusion
regi
on5
NSP
153
Non
stru
ctur
alRNA
bind
ing,
anta
goni
stof
inte
rfer
onre
spon
se6
VP6
45
DLP
(780
)Hyd
roph
obic
trim
er,g
roup
andsu
bgro
upan
tige
n7
NSP
334
Non
stru
ctur
alIm
portan
tfor
vira
lmRNA
tran
slat
ion,
PABP
hom
olog
ue,R
NA
bind
ing,
intera
ctswith
eIF4
G8
NSP
235
Non
stru
ctur
alIm
portan
tfor
geno
mere
plicat
ion/
pack
agin
g,m
ain
cons
titu
ento
fvirop
lasm
,NTPa
se,
RNA
bind
ing,
intera
ctswith
NSP
59
VP7
34Cleav
edsign
alse
quen
ce,h
igh
man
nose
glyc
osylat
ion
and
trim
min
gTLP
(780
)RER
integr
alm
embr
anegl
ycop
rotein
,ne
utra
lizat
ion
antige
n,Ca+
+bi
ndin
g10
NSP
420
Unc
leav
edsign
alse
quen
ce,h
igh
man
nose
glyc
osylat
ion
and
trim
min
gNon
stru
ctur
alRER
tran
smem
bran
egl
ycop
rote
in,
role
inm
orph
ogen
esis,v
iral
entero
toxi
n11
NSP
526
Phos
phor
ylated
,O-g
lyco
sylate
dNon
stru
ctur
alCon
stitue
ntof
viro
plas
m,int
erac
tswith
NSP
2,RNA
bind
ing,
Prot
ein
kina
se11
NSP
611
Non
stru
ctur
alCon
stitue
ntof
thevi
ropl
asm
,int
erac
tswithNSP
5
a Anu
mbe
rof
know
nfu
nction
alpr
oper
ties
wer
ead
ded,
man
yta
ken
from
Estes
2001
bM
olec
ular
weigh
tsba
sed
onap
pare
ntm
olec
ular
weigh
tsby
SDS-
PAGE
analys
is
-
Rotavirus Proteins: Structure and Assembly 195
Fig. 2ac X-ray structure of the VP8*, VP5*, and VP6. a X-ray
structures of VP5* andVP8* (shown in the backbone representation)
tted into the cryo-EM envelope of theVP4 spike derived from a 12-
resolution map. (Adapted from Dormitzer et al. 2004).b X-ray
structure of the VP6 trimer (monomers in red, green, and blue)
shown inribbon representation. (Mathieu et al. 2001). c Fitting of
the X-ray structure of the VP6trimers into the trimers around the
type I channel in the cryo-EM map of the DLP
studies of the rotavirus complexed with VP4-specic monoclonal
antibodies(Prasad et al. 1990; Tihova et al. 2001).
The VP4 spike exhibits a distinct structure with two distal
globular do-mains, a central body, and an internal globular domain
that is tucked insidethe VP7 layer in the peripentonal channel of
the T=13 icosahedral lattice(Shaw et al. 1993; Yeager et al. 1994).
X-ray structures of proteolytic frag-ments of VP4, VP8*, and VP5*
have been determined (Dormitzer et al. 2002,2004), and provide
strong evidence that the distal globular domain of the VP4spike is
composed of VP8* with the remaining body of the spike consisting
ofVP5* (Fig. 2a). The crystallographic studies on VP5*, as
discussed in connec-tion with cell entry below, have also indicated
the possibility of an alternateoligomerization state of VP4
(Dormitzer et al. 2004).
3.2Aqueous Channels
One of the distinctive features of the rotavirus architecture is
the presence oflarge channels that penetrate through theVP7 andVP6
layers. These channelsallow for the passage of aqueous materials
and biochemical substrates into
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196 J. B. Pesavento et al.
and out of the capsid. The 132 channels at the ve-fold and quasi
six-foldpositions of the T=13 lattice are grouped into three
distinct types. Twelvetype I channels are located at the ve-fold
vertices of the capsid (arrows,Fig. 1b). There are 60 type II
channels at each of the pentavalent locationssurrounding the type I
channels, near which VP4 is attached to VP7 and VP6(Fig. 1b). The
60 type III channels are located at the remaining
hexavalentpositions on the capsid surrounding the icosahedral
three-fold axes (Fig. 1b).
3.3VP6 Layer
The intermediate layer is formed by the VP6 protein, and is in
direct contactwith the VP7 later. Particles carrying VP6 on the
outside are called double-layered particles (DLPs). The VP6 layer
maintains the same icosahedral sym-metry as the VP7 layer with 780
copies of VP6 arranged as 260 trimers ona T=13 icosahedral lattice
(Fig. 1c). These trimers are located right below theVP7 trimers
such that the channels in the VP7 and VP6 layers are in regis-ter.
The DLP is the transcriptionally competent form of the virus during
thereplication cycle. VP6 is the major protein of the rotavirus
particle by weight.It plays a key role in the overall organization
of the rotavirus architecture byinteracting with the outer layer
proteins, VP7 and VP4, and the inner mostlayer protein VP2. Thus,
it may integrate two principal functions of the virus:cell entry
(outer layer) and endogenous transcription (inner layer). The
X-raystructure of VP6 has been determined and it shows that VP6 has
two domains(Fig. 2b, c) (Mathieu et al. 2001). It its overall
structure, VP6 is similar to theVP7 of BTV (Grimes et al. 1997,
1998) and to the 1 protein of orthoreovirus(Liemann et al. 2002).
The distal domain with an eight-stranded antiparallel-sandwich
foldmakes contactwith theVP7 layer, and the lower domain,
con-sisting of a cluster of-helices,makes contact with the innerVP2
layer. Fittingof the X-ray structure of VP6 into the cryo-EM
structure of the DLP showsthat the VP6 trimers interact laterally
to form the T=13 layer (Mathieu et al.2001). There appear to be two
types of contacts between the trimers. The con-tacts, across the
quasi two-fold axes and closer to the icosahedral three-foldaxis
are similar, whereas the contacts are varied as the trimers
approach theicosahedral ve-fold axis. In contrast to VP7 of BTV
(Grimes et al. 1998), theVP6 trimer exhibits extensive lateral
interactions involving charged residues.
3.4VP2 Layer and Transcription Enzyme Complex
Underneath the VP6 layer is the innermost protein layer of the
rotavirusstructure. The particle structure at this level is
referred to as the single-layer
-
Rotavirus Proteins: Structure and Assembly 197
particle (SLP). The SLP houses the dsRNA genome within a protein
layercomposed of 120 copies of VP2 (Fig. 3a) arranged in an unusual
T=1 icosahe-dral lattice with two molecules in the icosahedral
asymmetric unit (Lawtonet al. 1997b). All the structurally
characterized members of the Reoviridaeand of other dsRNA viruses
such as phi6 and LA viruses exhibit this unique
Fig. 3ae Structural organization of the VP2 layer, genomic
dsRNA, and transcriptionby the rotavirus DLP. a Surface
representation of the outer portion of VP2. In oneof the 60 dimers
that constitute this layer, the VP2 subunits are colored in red
andpurple to indicate their orientations and connections to one
another. (Adapted fromLawton et al. 1997b). b Cut-away view of the
DLP. The VP6 and VP2 layers werepeeled halfway to expose the
outermost layer of genomic organization. The outerlayer of RNA has
a dodecahedral appearance and surrounds each of the
VP1VP3star-shaped complexes at the ve-fold vertices. (Adapted from
Prasad et al. 1996).c Model for genome organization around the
VP1/3 transcription enzyme complex.The outer green portions
represent a cut-away view of the VP2 layer. The yellow
spiralsindicate dsRNA gene segments and the red spheres represent
the VP1/3 transcriptioncomplexes. (Adapted from Pesavento et al.
2003). d A DLP is shown with mRNAtranscripts exitingoutby
theproposedpathway through the type I channel at ave-foldvertex.
The transcripts are colored as gray strands. e Close-up view of a
transcribingDLP. The pink bowling-pin-shaped density is the result
of the exiting transcript seenin the reconstructions of actively
transcribing DLPs. (Lawton et al. 1997a)
-
198 J. B. Pesavento et al.
organization of the core protein (reviewed in Prasad and
Prevelige 2003). Thestructural organization of the corresponding
layers in three of the ReoviridaemembersBTV (Grimes et al. 1998),
orthoreovirus (Reinisch et al. 2000), andrice dwarf virus (Nakagawa
et al. 2003)have been visualized at the atomiclevel. From the X-ray
structure of the BTV core particle, which closely resem-bles the
rotavirus DLP, Grimes et al. (1998) have argued that the
pentamericcaps ofVP3 (equivalent of rotavirusVP2) dimers are
building blocks in the as-sembly of this layer. VP2 expressed using
the baculovirus expression system,forms helix-like structures that
can form spherical particles at lower concen-trations (Zeng et al.
1994) and co-expression of VP2 with VP1 and/or VP3results in the
self-assembly of these proteins into VP1/2, VP2/3, and
VP1/2/3virus-like particles (VLPs) (Wentz et al. 1996). Comparative
cryo-EM analysisof these particles showed that 12 copies of the
VP1/VP3 transcription enzymecomplexes are attached to the inner
surface of theVP2 layer at each of the ve-fold vertices of the SLP
and surrounding each of the transcription complexesis genomic dsRNA
(Fig. 1c). Similar structural localization of the
enzymes,particularly the polymerase, required for endogenous
transcription is foundin other members of the Reoviridae such as
BTV (Gouet et al. 1999; Nasonet al. 2004), rice dwarf virus
(Nakagawa et al. 2003; Zhou et al. 2001), aquare-ovirus (Nason et
al. 2000), orthoreoviruses (Zhang et al. 2003), and cypovirus(Zhang
et al. 1999). Such structural conservation is not surprising given
thatin all these viruses endogenous transcription of multiple
segments is a com-mon and necessary phenomenon. However, a
contrasting feature is in regardto the location of the capping
enzyme. In viruses such as rotavirus, BTV, andrice dwarf virus, the
capping enzyme is suggested to be inside the core layer,whereas in
viruses such as the orthoreovirus, aquareovirus, and cypovirus,the
capping enzyme forms a distinctive turret structure with a central
holelocalized at the virion ve-fold axis (Hill et al. 1999).
3.5Genome Organization
The question of how the dsRNA segments are arranged inside the
capsidis particularly interesting considering that they are
transcribed simultane-ously and repeatedly within the connes of the
capsid. By analyzing thestructural differences between empty
virus-like particles (VLPs) and nativerotavirus particles, Prasad
et al. (1996) showed that a signicant portion of thegenome is
statistically ordered and manifests as concentric layers of
densityinside the icosahedrally averaged reconstructions of the
rotavirus particles(Fig. 3b). Similar structural manifestation of
the genome is indeed seen inthe X-ray structure of the BTV core and
cryo-EM reconstructions of several
-
Rotavirus Proteins: Structure and Assembly 199
other dsRNA viruses. However, because of the implicit use of
icosahedralsymmetry averaging in the structure determination of
these viruses, eitherby crystallography and or cryo-EM, the precise
organization of the individualgenome segments is lost.
Interestingly, in rotavirus, using a combination ofbiochemical and
cryo-EM techniques, Pesavento et al. (2001) showed thatthe
rotavirus genome can undergo reversible condensation and
expansionwithout affecting the integrity of the surrounding capsid
layers. A plausiblemodel that emerges from the available
biochemical and structural data forrotaviruses and other dsRNA
viruses, is that each genome segment is spooledaround a
transcription complex (consisting of VP1 and VP3) that is
anchoredto the inner surface of the VP2 layer at the ve-fold axis
(Gouet et al. 1999;Pesavento et al. 2003b). Such amodel (Fig. 3c)
allows for up to 12 independenttranscription complexes, each
associated with an individual dsRNA segmentfor concurrent
transcription.
4Reassortants
Although most of the cryo-EM structural studies have been
performed ona fewselected strainsof rotavirus, these studies
clearly indicate that thegeneralarchitectural features are
generalizable and independent of the strains. Cryo-EM structural
studies have been reported on several rotavirus reassortants.These
structural studies indicate that the capsid structure remains
unalteredexcept for the VP4 spikes. Rotavirus reassortment occurs
widely in natureand represents a major force for genetic diversity
along with point mutationsand gene rearrangements (Desselberger
1996; Iturriza-Gomara et al. 2001).The structures of reassortants
show that while VP4 generally maintains theparental structure, when
moved to a heterologous protein background, incertain reassortants
there are subtle alterations in the conformation of VP4(Pesavento
et al. 2003a). The alterations in the VP4 conformation
correlatedwith the observation of unexpected VP4-associated
phenotypes. Interactionsbetween heterologous VP4 and VP7 in
reassortants expressing unexpectedphenotypes appear to induce the
conformational alterations seen in VP4.
5Protease-Enhanced Infectivity
From their locations in the structure of rotavirus, VP7 and VP4
are obviouscandidates to be implicated in the cell entry processes.
Although early studies
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200 J. B. Pesavento et al.
implicated VP7 in the cell entry process (Fukuhara et al. 1988;
Sabara et al.1985), subsequent studies have increasingly indicated
the involvement of VP4not only in cell attachment and cell
penetration, but also in hemagglutina-tion, neutralization,
virulence, and host range (Burns et al. 1988; Fiore et al.1991;
Kirkwood et al. 1998; Lopez et al. 1985; Ludert et al. 1996, 1998;
Mackowet al. 1988). Prior to its interaction with the host cell,
VP4 is proteolyticallycleaved for efcient internalization of
rotaviruses into cells. This is particu-larly relevant considering
that rotavirus replication takes place in enterocytesin the small
intestine, an environment rich in proteases. Proteolytic cleav-age
of VP4 enhances viral infectivity by several fold (Arias et al.
1996; Esteset al. 1981) and facilitates virus entry into cells
(Kaljot et al. 1988). Proteolysisof VP4 generates two fragments,
VP8* (aa 1247) and VP5* (248776) andthese fragments remain
associated with the virion (Fiore et al. 1991; Lopezet al. 1985).
Trypsinized viruses enter cells more efciently without using
theendosomal pathway, compared to particles that are not
trypsinized (Kaljotet al. 1988; Keljo et al. 1988). In vitro
experiments have shown that prote-olytically activated particles,
as well as recombinant VP5*, possess lipophilicactivity (Dowling et
al. 2000; Nandi et al. 1992; Ruiz et al. 1994). Althoughrotavirus
is a nonenveloped virus, it is interesting to note some parallels
be-tween rotavirusVP4 and cell attachment proteins in enveloped
viruses such asinuenza viruses. Proteolytic cleavage is as
essential for infection in inuenzavirus as it is for rotavirus,
because it primes the HA (hemagglutinin) pro-tein for an ensuing
irreversible conformational change, which occurs in thelow-pH
environment of endosomes prior to membrane fusion. The
rotavirusVP5* andVP8* trypsin cleavage products are analogous to
the proteolyticallycleaved fragments of the inuenza virus
hemagglutinin, HA1 and HA2. Muchlike rotavirus VP8*, the HA1
subunit plays an accessory role by providinginitial binding to the
cell via sialic acid containing receptors. HA2 functionsmore
likeVP5*, as it is required and sufcient on its own for cell fusion
(Wileyand Skehel 1987).
5.1Trypsin-Induced Unique Order-to-Disorder Transition in the
Spike
The molecular mechanism of increased infectivity by proteolysis
is not wellunderstood. To understand the structural basis of
trypsin-enhanced infec-tivity in rotaviruses, Crawford et al.
(2001) examined the biochemical andstructural properties of
rotaviruses grown in the absence (nontrypsinizedrotavirus, NTR) or
presence (trypsinized rotavirus, TR) of trypsin. The infec-tivity
of the NTR particles is drastically reduced, as anticipated.
Exogenousaddition of trypsin toNTRparticles increased their
infectivity but to nowhere
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Rotavirus Proteins: Structure and Assembly 201
near the level of infectivity seen with TR particles. Despite
clear biochemi-cal indications for the presence of uncleaved VP4 in
correct stoichiometricproportion in the NTR particles, the spikes
in the cryo-EM reconstruction ofthese particles are not visualized
in contrast to thewell dened spike structureseen in the particles
that are grown in the presence of trypsin (Fig. 4a , b).
Fig. 4ad Effects of trypsin and pH on the spike structure. The
highly exible VP4spike protein on rotavirus assumes altered
conformations due to proteolytic cleavageor encountering high pH. a
Rotavirus grown in the absence of trypsin (upper panel)has low
infectivity and theVP4 spike is disordered on particles (i.e., not
represented incryo-EM reconstructions). (Crawford et al. 2001). b
Proteolytically cleaved rotavirushas high infectivity and a
well-ordered spike appearing dimeric at the top. c Treatmentof
rotaviruswith~pH11 induces a conformational change in the spike
resulting ina tri-lobed stunted spike and unmasks a cell binding
domain that appears to be involved ininfectionof cells by a sialic
acid-independentmechanism. (Pesavento et al.
2005).dThehigh-pH-altered short spikes are recognized byVP5*-specic
2G4-Fab fragments, andthree Fab fragments are seen binding to each
altered spike (Pesavento et al. 2005)
-
202 J. B. Pesavento et al.
These results thus indicate that trypsin cleavage imparts
structural order tothe VP4 spikes on de novo synthesized virus
particles and that these orderedspikes make virus entry into cells
more efcient (Crawford et al. 2001).
The ideaofa trypsin-induceddisorder-to-order transition is
indeeduniqueand has not been documented with any other virus thus
far. Does trypsin actfrom within or outside of cells? One
possibility is that during virus infection,trypsin acts outside
cells on the newly formed VP4 and that this trypsinizedVP4 is able
to assemble properly onto the rotavirus particles. This
hypothesisis consistent with the nding, using confocal microscopy
of virus-infectedMA104 cells, that high amounts of VP4 are present
at the plasma membraneapproximately 3 h after infection and that
the N-terminal region, i.e., VP8*,is accessible to antibodies
(Nejmeddine et al. 2000). Similar results wereobtained with cells
transfected with a VP4 plasmid, suggesting that
VP4targetingdependsonsignals in theprotein rather thanon
thepresenceof virusparticles. Targeting of VP4 to the plasma
membrane appears to be a generalphenomenon as it is seen in both
polarized and nonpolarized cells (Sapinet al. 2002). Further
structural and biochemical studies are needed to providea better
understanding of how and where trypsin affects spike assembly.
6Cell Entry
The consensus opinion that has emerged from several recent
studies is thatrotavirus cell entry is a coordinated multistep
process involving sequentialinteractions with sialic acid (SA)
-containing receptors in the initial cell at-tachment step. Next,
interactions are throught to occur with hsp70, and inte-grins such
as v3, 41, 21 during the subsequent postattachment steps(reviewed
in Lopez and Arias 2004). In the entry process, the VP8* domainis
involved in the interactions with SA, whereas VP5* is implicated in
theinteractions with integrins. Involvement of VP8* in cell
attachment is fur-ther supported by studies that show that several
VP8*-specic neutralizingmAbs block cell attachment. The X-ray
structure of the VP8*SA complexhas shown that VP8* has a
beta-sandwich fold similar to that of galectins,whose natural
ligands are carbohydrates (Dormitzer et al. 2002). The SAbindsto a
shallow pocket between the two -sheets, a region that is distinct
fromthe carbohydrate binding pocket in the galectins, which is
blocked in theVP8*. Involvement of SA during rotavirus infections
is not an essential stepin all rotavirus strains. For many of the
rotavirus strains, including humanrotaviruses, cell entry is
SA-independent (Ciarlet et al. 2001). In these viruses,the majority
of neutralizing mAbs select mutations in VP5* (Kirkwood et al.
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Rotavirus Proteins: Structure and Assembly 203
1996, 1998; Padilla-Noriega et al. 1995), suggesting that cell
entry is mediatedmainly by the VP5*. An interesting question is
what the role of VP8* mightbe in these SA-independent viruses.
6.1Possible Structural Alterations in VP4 During Cell Entry
How does VP4 facilitate such multistep entry processes in
rotavirus? It is pos-sible that VP4 undergoes distinct
conformational changes at various stagesduring cell entry to mask
certain epitopes and reveal others in order to op-timally interact
with different receptors and the cellular membrane. Suchdistinct
conformational states during cell entry processes have been
observedin viruses such as inuenza virus (Bullough et al. 1994),
avivirus (Modiset al. 2004; Mukhopadhyay et al. 2003), alphavirus
(Gibbons et al. 2004) andpicornaviruses (Belnap et al. 2000).
Recent studies on rotavirus clearly pointto conformational changes
of VP4 during cell entry. In addition to the dras-tic
conformational change from a exible to a rigid-bilobed spike
structureupon trypsinization, as discussed above (Crawford et al.
2001), recent X-raycrystallographic studies of VP5* (Dormitzer et
al. 2004) and cryo-EM stud-ies in high-pH-treated rotaviruses
suggest the possibility of further structuralchanges in the spike
structure thatmay be relevant during rotavirus cell entry.
6.1.1Is the VP4 Spike a Trimer?
In the crystal structure, VP5* is a trimerwith substantial
intersubunit interac-tions (Dormitzer et al. 2004). That is, by
itself, VP5* has a propensity to formstrong trimers. Why, then, in
the cryo-EM structures is the spike a dimericstructure? Two
individual monomers of VP5* clearly t into the main body ofthe
spike in the cryo-EMstructure (Fig. 2a). Aproposedpossibility is
that eachspike is indeed a trimer of VP4, and upon trypsinization,
two of them formthe visible spike, as seen in the cryo-EM
reconstruction of the trypsinized ro-tavirus particles, with the
other monomer being oppy and not visible in thereconstruction
(Dormitzer et al. 2004). During cell entry, by a yet
unknownentry-associated event, the oppy VP4 monomer together with
the other twomolecules, trimerizes as seen in the VP5* crystal
structure.
6.1.2pH-Induced Changes of the Spike:Implication for Cell Entry
and Antibody Neutralization
Recent studies on high-pH-treated rotavirus have uncovered an
interestingphenomenon that appears to substantiate the above
proposal (Pesavento et al.
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204 J. B. Pesavento et al.
2005). At elevated pH, the spike undergoes a drastic
irreversible conforma-tional change and becomes stunted with a
pronounced tri-lobed appearance(Fig. 4c). Biochemical analysis of
pH-treated particles indicates that VP4 ispresent in the same
amount as in native particles. Three Fab fragments ofthe
VP5*-specic neutralizing monoclonal antibody, 2G4, are seen to bind
tothe altered spike structure (Fig. 4d). One strong possibility
from these ob-servations is that VP4 has undergone a dimer to
trimer transition. Despitethe loss of infectivity and the ability
to hemagglutinate, the high-pH-treatedparticles surprisingly
exhibit SA-independent cell binding, in contrast to na-tive
virions, which exhibit SA-dependent cell binding. These studies
have alsoshown that the binding of 2G4-Fab to native particles
completely protectsthe spikes from undergoing pH-induced
conformational changes and pre-serves the SA-dependent cell binding
and hemagglutinating functions of thevirion. However, when 2G4 is
bound to the pH-altered particles, cell bindingis completely lost.
A hypothesis that emerges from this study is that high-pHtreatment
triggers a conformational change thatmimics apost-SAattachmentstep
to expose an epitope recognized by one of the downstream receptors
inthe rotavirus cell entryprocess, and themechanismbywhich
the2G4antibodyneutralizes infectivity is by preventing this
conformational change.
In their cell attachment, the pH-treated particles appear to
resemble thenar3 mutant of rhesus rotavirus (RRV) (Graham et al.
2003; Zarate et al.2000a). This mutant exhibits SA-independent cell
binding in contrast to itsparental strain and has been shown to
attach to the cell surface by inter-acting with integrin 21 through
the DGE motif in VP5*. As in the high-pH-treated particles, 2G4
antibody binding to the nar3 mutant inhibits cellbinding (Zarate et
al. 2000b). A distinct possibility is that the DGE motif(residues
308310) becomes exposed in the pH-treated particles, and the2G4-Fab
inhibits cell binding of the pH-treated particles by sterically
hinder-ing the accessibility of this motif. In the studies by
Pesavento et al. (2005), pHwas used to trigger the conformational
changes. During a natural infectionprocess, it is not known what
triggers the conformational changes necessaryto interact with
downstream receptors. As yet there are no structural
studiesreported of rotavirus complexed with any of the multiple,
proposed receptorsmolecules.
7Endogenous Transcription
The next stage in the replication cycle of the virus is the
transcription ofdsRNA segments into viable mRNA molecules that can
be processed for
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Rotavirus Proteins: Structure and Assembly 205
template generation and viral protein production. During the
process ofcell entry, the outer layer is removed and the resulting
DLPs in the cyto-plasm become transcriptionally competent (Estes et
al. 2001). The dsRNAsegments are transcribed within the structural
connes of the DLP. Cryo-EM structural studies have shown that DLPs
remain structurally intact dur-ing the process of transcription,
and the nascent transcripts exit throughthe type I channels that
penetrate the inner VP2 and outer VP6 capsid lay-ers of the DLP at
the ve-fold vertices (Fig. 3d) (Lawton et al. 1997a). TheDLP
possesses the complete enzymatic activities needed to synthesize
notonly mRNA transcripts but also to properly guanylate and
methylate thecap structure at the 5 end of each mRNA to facilitate
translation by thecellular translation machinery. These enzymatic
functions are carried outby VP1, the RNA-dependent-RNA polymerase
(Valenzuela et al. 1991), andVP3, a guanylyltransferase and
methyltransferase (Chen et al. 1999). WhileDLPs are
transcriptionally competent both in vitro and in vivo, the TLPs
aretranscriptionally incompetent. Certain monoclonal antibodies,
which bindto the distal end of VP6, almost 140 away from the site
of transcriptioninitiation, inhibit transcription (Ginn et al.
1992; Kohli et al. 1993; Thouveninet al. 2001). From cryo-EM
studies of DLPs complexed with these antibodies,it has been
proposed that binding of ligand, such as an antibody or VP7,induces
a conformation change at the interface of the VP2 and VP6 layersto
inhibit sustained elongation and translocation of the transcripts
(Lawtonet al. 1999). Further higher-resolution structural analysis
of TLPs and DLPsis necessary to understand the structural basis of
transcriptional activationand inhibition.
8Genome Replication and Packaging
Following endogenous transcription and release of the
transcripts, the ro-tavirus replication cycle may be viewed as
having three subsequent majorstages: (1) translation and synthesis
of the viral proteins; (2) replication,genomepackaging, andDLP
assembly; (3) budding of the newly formedDLPsinto the ER and
assembly of the outer layer to form mature TLPs (reviewedin Estes
2001). The positive-stranded RNA transcripts encode the
rotaviralproteins and function as templates for production of
negative strands tomakethe progeny dsRNA. Recent studies with siRNA
have indicated that there arelikely to be two separate pools of
mRNA for these distinct functions (Silvestriet al. 2004).
-
206 J. B. Pesavento et al.
8.1NSP3 and Genome Translation
The nonstructural protein NSP3 is implicated in the specic
recognition ofthe rotaviral mRNAs and in facilitating their
translation using the cellularmachinery (Piron et al. 1998, 1999;
Vende et al. 2000). NSP3 is a functionalhomologue of cellular
poly(A) binding protein (PABP). While the N-terminaldomain of NSP3
interacts with the 3-consensus sequence of the rotaviral
viralmRNAs, the C-terminal domain interacts with eIF4G to enable
circularizationof viral mRNA and its delivery to the ribosomes for
viral protein synthesis.The X-ray structures of both the N-terminal
domain complexed with theconsensus rotaviral mRNA sequence, and
that of the C-terminal domainbound to a peptide that corresponds to
the binding site on eIF4G have beendetermined (Deo et al. 2002;
Groft and Burley 2002). These studies clearlyindicate that NSP3
functions as a homodimer. Both the domains have novelfolds. While
the RNA binding domain forms a heart-shaped asymmetricdimer, the
C-terminal domain forms a rod-shaped symmetric dimer.
ThedimericN-terminaldomain tightlybinds to the consensus3-endof
themRNAinside a tunnel formed at the dimeric interface. The binding
of NSP3 to themRNA had also been proposed as a possible mechanism
to transport newlymade mRNAs to viroplasms for subsequent
replication.
8.2NSP2 and NSP5
Replication, genomepackaging and assembly of theDLPoccur in
perinuclear,nonmembrane-bound, electrondense inclusions called
viroplasms,which ap-pear 23 h after infection. Several in vivo and
in vitro studies have stronglyimplicated two of the nonstructural
proteins NSP2 and NSP5, not only inthe formation of the viroplasm,
but also in genome replication and packag-ing (Afrikanova 1998;
Aponte et al. 1996; Gallegos and Patton 1989; Kattouraet al. 1994;
Petrie et al. 1984). Co-expression of NSP2 and NSP5 in
uninfectedcells form viroplasm-like structures (Fabbretti et al.
1999). NSP5 is a dimericphosphoprotein rich in Ser and Thr residues
that undergoes O-linked gly-cosylation (Afrikanova et al. 1996;
Poncet et al. 1997) . In co-transfectionexperiments with NSP5 and
NSP2, NSP2 has been shown to upregulate phos-phorylation of NSP5
(Afrikanova 1998). In vivo studies have shown that thesetwo
proteins along with VP1, the viral RNA polymerase, are co-localized
inthe viroplasms and that they are themain constituents of the
replication inter-mediates (reviewed in Taraporewala and Patton
2004). Further evidence forthe involvement of NSP2 and NSP5 in the
formation of viroplasms, genomereplication, and virion assembly is
provided by recent studies using siRNA
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Rotavirus Proteins: Structure and Assembly 207
techniques, which showed that suppression of eitherNSP2 orNSP5
expressioninhibits the formation of viroplasms, genome replication,
and viral assem-bly (Campagna et al. 2005; Silvestri et al. 2004).
Noting that the viral mRNAlocated outside the viroplasms that are
involved in translation are suscepti-ble to siRNA-induced
degradation, while the mRNA in the viroplasms thatundergo
replication are not, Silvestri et al. (2004) have suggested that
thetranscriptionally active progeny DLPs form foci for the
formation of the viro-plasms, thus eliminating the necessity for
two spatially distinct locations fortranscription and replication.
This model eliminates the necessity of havingto transport viral
mRNAs and viral proteins, as per an earlier model, to theviroplasms
for negative strand synthesis and subsequent DLP assembly andgenome
packaging.
Biochemical studies on recombinantNSP2have shown that it readily
formsan octamer andhasNTPase (nucleotide triphosphatase),
ssRNA-binding, andhelix destabilizing activities (Taraporewala et
al. 1999, 2001; Taraporewala andPatton 2001). Based on these
properties, it has been suggested that NSP2 mayfunction as a
molecular motor using the energy derived from NTP hydrolysisto
facilitate genome packaging. The X-ray structure of NSP2 has
providedsome insights into the locations of NTP and RNA binding
sites (Jayaram et al.2002). NSP2 is a two-domain / protein. The two
domains are separatedby a deep cleft. The N-terminal domain is
predominantly -helical with onlya few -strands, whereas the
C-terminal domain has a twisted antiparallel -sheet with anking
-helices. Despite any detectable sequence similarity,
thepolypeptide fold in this domain is highly similar to that
observed in the HIT(histidine triad) family of nucleotidyl
hydrolases (Lima 1997). Based on thissimilarity, it was suggested
that this domain contains theNTP binding pocket.Recent mutational
analysis based on the structural observations is consistentwith
such a prediction (Carpio et al. 2004). NSP2 forms a
doughnut-shapedoctamer with a 35--wide central hole, and four
grooves related by a four-foldaxis on the sides of the octamer.
These grooves, lined with basic residues, aresuggested to be the
sites for RNA binding. Thus while the NTPase activityis localized
in the monomeric subunit, the ability to bind RNA and otherproteins
such as NSP5 and VP1 may require the formation of the octamer.
Based on the structure of NSP2 and its functional properties, it
is tempt-ing to speculate that the replication complex is organized
around the NSP2octamer providing a platform or a scaffold (Jayaram
et al. 2004). It is possiblethat the hydrophobic side of the
octamer, around the four-fold axis, may bindto VP1; given that NSP5
is an acidic protein, the basic grooves of the NSP2octamer may be
the binding sites for NSP5. Although the role of NSP5 inthe overall
replication process remains to be elucidated, it is plausible that
byhaving its binding site on NSP2 overlap with that of the RNA
binding site, the
-
208 J. B. Pesavento et al.
function of NSP5 is to regulate the binding of RNA by NSP2
during replica-tion and packaging. It is still unclear whether
NSP6, which is encoded by analternating open reading frame in the
gene segment 11 along with NSP5 andis also present in the
viroplasms, has any role in genome replication and/orpackaging.
NSP6 interacts with NSP5 and it is suggested that it might havea
regulatory role in the self-association of NSP5 (Torres-Vega et al.
2000).
8.3A Working Model for Genome Encapsidation in Rotavirus
How the correct set of 11 segments of dsRNAget encapsidated into
each virionremains entirelyunclear.Given thatmultiple segmentsof
varied lengthhave tobe encapsidated, and that eachonehas
tooccupydifferent vertices to associatewith a transcription enzyme
complex, as per the current model of genomeorganization, it is
unlikely that the dsRNAgenome segments are encapsidatedinto
preformed empty capsids as in some of the bacteriophages. Instead,
theencapsidation could be concurrent with the capsid assembly as
proposed byPesavento et al. (2003). In thismodel (Fig. 5), the
capsid assembly begins withthe association of 12 units, each unit
consisting of pentamers of VP2 dimersin complex with a
transcription enzyme complex (VP1/VP3) and a genomesegment, to form
the SLP and provide a scaffold for the subsequent assembly
Fig. 5 Aworkingmodel for genome encapsidation in rotavirus.
Based on the availablebiochemical and structural data, one possible
model for genome encapsidation isshown. All the components that are
likely to be involved in this process are indicated.See Sect. 8.3
in the text for details
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Rotavirus Proteins: Structure and Assembly 209
of the VP6 trimers leading to the assembly of a DLP. The
proteinaceousparts of each of these units may represent the
replicase complex in whichmRNA, brought in with the aid of
nonstructural proteins (NSP2/NSP5), isfed into the enzyme complex
for the synthesis of the negative strand and theformationof
theduplexRNA,whichgets spooledaround theenzymecomplex.In such a
process, NSP5may function as an adapter, with its ability to
interactwith VP2 and to facilitate interactions between NSP2 and
the VP1VP3VP2complex (Berois et al. 2003). This model raises an
important question as tohow a correct set of 11 (as in rotavirus)
distinct segments is brought together.It is possible that specic
RNARNA interactions coordinate this process.
9Maturation and Release
Maturation and release represent the nal steps of the rotavirus
replicationcycle.Once formed,DLPsbud from the viroplasms into
theproximally locatedER (Estes 2001), and by a mechanism that is
not clear DLPs acquire the outerlayer consisting of VP7 and VP4.
This budding process is facilitated by thenonstructural proteins
NSP4, which has a binding site for VP6 (Au et al.1989, 1993; Meyer
et al. 1989; Tian et al. 1996). Both NSP4 and the outerlayer
protein VP7 are synthesized on the ER-associated ribosomes and
co-translationally inserted into the ER membrane. NSP4 is a
predominantly-helical glycoprotein that forms a tetramer with its
C-terminal 131 residueson the cytoplasmic side of the ER. The
C-terminal residues form a bindingsite for VP6 (OBrien et al. 2000;
Taylor et al. 1992, 1993). As yet there isno structural information
on NSP4, except that of a small region that isresponsible for
tetramerization (Bowman et al. 2000). Recent studies usingRNA
interference have shown that accumulation of rotaviral proteins
andindeed, DLPs and TLPs, are blocked by silencing the expression
of the NSP4gene (Lopez et al. 2005). This result indicates that
NSP4 may have previouslyunexpected functions related to virus
maturation. Aside from its role in viralmorphogenesis, NSP4 is a
viral enterotoxin capable of inducing diarrhea onits own in mice
(Ball et al. 1996; Estes 2001, 2003; Sasaki et al. 2001).
During the buddingprocess,DLPs get enveloped transiently in
theER. Thismay be an intermediate stage during acquisition of the
VP7 layer. Silencingthe expression of VP7 does not affect the
assembly of DLPs but leads to theaccumulation of enveloped DLPs in
the ER, thereby suggesting that VP7 isrequired for removal of the
lipid envelope (Lopez et al. 2005). Although theassembly of the VP7
layer onto the DLPs, as generally agreed, takes place inthe ER,
where and how the spike protein VP4, which is synthesized on
free
-
210 J. B. Pesavento et al.
cytosolic ribosomes, is assembled onto the particles is unclear.
The cryo-EMstructure of the particles produced by silencing the VP4
gene during virusinfection clearly shows all the features of the
native TLP structure except forthe VP4 spikes (Arias et al. 2004;
Dector et al. 2002). These results suggestthat neither the proper
assembly of VP7 nor the budding of the DLPs into theER require VP4.
Based on the results that indicate VP4 alone can trafc to theplasma
membrane of the infected cells (Nejmeddine et al. 2000; Sapin et
al.2002), a likely possibility is that assembly of VP4 onto viral
particles may takeplace at the plasma membrane shortly before
particle release and that VP4may be involved in the early stages of
virus release. The presence of trypsinor a protease outside of
cells may access the VP4 and bring about appropriatestructural
alterations for its proper assembly on the particles with the
VP7layer already assembled.
10Conclusion
In last few years, there has been tremendous progress in our
understanding ofthe structural and biochemical aspects of a variety
of themolecular processesinvolved in rotavirusmorphogenesis,
includingprotease enhanced infectivity,cell entry, antibody
neutralization, genome replication, and maturation. Thishas
beenmadepossible by the appropriate use of structural techniques
such ascryo-EM and X-ray crystallography either independently or in
combination.Particularly noteworthy are the insights provided by
the atomic structures ofseveral of the rotaviral proteins,
including VP4, VP6, NSP3, NSP4, and NSP2.In parallel, this progress
was facilitated by equally important advances inthe molecular
biology of rotaviruses, resulting in recombinant proteins
andvirus-like particles, along with the successful application of
RNA interferencetechniques. These studies have uncovered several
unique aspects of rotavirusmorphogenesis and as always raise
several intriguing new questions aboutthese viruses such as:
1. How and where does the assembly of VP4 take place in infected
cells?
2. How does trypsin facilitate proper assembly of the VP4
spike?
3. How does VP4 facilitate interactions with the variety of
proposed recep-tors?
4. How is the endogenous transcription controlled by the
addition or theremoval of the outer capsid layer?
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Rotavirus Proteins: Structure and Assembly 211
5. How is the process of genome replication, packaging and
assembly or-chestrated and controlled by the interplay between
structural and non-structural proteins?
6. What is the structural and molecular basis of NSP4 function
both inrelation to viral pathogenesis and morphogenesis?
Dissecting the rotavirus functions in terms of its individual
proteins wouldhave been much easier if a reverse genetics system
was available. Given thecomplexityof this virus, or
anyothermemberof theReoviridae for thatmatter,establishing such a
system is indeed a daunting task. A major achievementin the near
future, as a result of continued and better understanding of
theprocesses that control rotavirus morphogenesis, could be the
establishmentof a reverse genetics system.
Acknowledgements This work was supported by NIH grants AI-36040
(B.V.V.P.) andDK-30144 (M.K.E.) and a grant from R. Welch
foundation (B.V.V.P). J.B.P. acknowl-edges the support of NSF
training grant BIR-9256580.
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